CN106796164A - The blood platelet targeting microfluidic separation of cell - Google Patents

Abstract

The application is related to for from sample fluid, such as biofluid, what such as blood, marrow, pleural effusion were related to blood platelet is separated in ascites has core target cell, for example, circulate the method and system of epithelial cell, circulating tumor cell (CTC), circulating endothelial cells (CEC), circulating stem cell (CSC), neutrophil leucocyte and macrophage.The method includes obtaining the cell capture chamber of the multiple bound fractions containing one or more walls for being bound to the room, and the wherein bound fraction is combined with blood-platelet specific；Any blood platelet in making bound fraction and sample it is related have core target cell combine under conditions of make sample fluid stream cross cell capture chamber to form compound；And from the compound separate to collect blood platelet it is related have core target cell.

Description

The blood platelet targeting microfluidic separation of cell

Cross-Reference to Related Applications

This application claims the priority of the U.S.Provisional Serial 62/034,522 of submission on the 7th of August in 2014, its is complete Portion's content is incorporated by reference into the application.

Statement on federal funding research

The present invention is that the numbering provided in NIH is 5-U01-EB012493-04 and EB002503- 06A1, and American Cancer Society provide numbering be 125929-PF-14-137-01-CCE government-funded under carry out.Political affairs Mansion has certain rights in the invention.

Technical field

The present invention relates to separate karyocyte from fluid such as blood.

Background technology

Transfer, diffusion and growth of the tumour cell from original site to remote organ represent cancer most destructiveness and cause The attribute of dead property, and the reason for be 90% cancer mortality.Although not yet setting up the system understanding to shifting biology, more To recognize importance of the circulating tumor cell (CTC) as transfer initiator cell more, it will be the early stage of cancer progression Diagnosis, sign and monitoring provide potential convenient source.However, being reliably detected from the blood of cancer patient and Noninvasive Separating CTC and other karyocytes still technically has challenge, is not only because them and exists extremely rarely (as little as Have one in 1,000,000,000 or more haemocyte), and it is also due to the heterogeneous water of biophysics and biochemical property It is flat.

For size, CTC and other rare karyocytes can be as small as 5 microns to 8 microns, the chi of such as red blood cell It is very little, or 8 microns to 18 microns, it substantially has same size with HL, or is more than 100 microns in CTC cluster forms. For surface chemistry, the biology for targetting CTC and epithelial cell in positive selection method under normal conditions is widely used in The expression of label epithelial cell adhesion molecule (EpCAM) has shown that larger difference between clinical sample, and as upper The result of skin-mesenchymal transformation (EMT) its can also significantly be lowered with cancer progression.And, have reported tumour cell and turning There is active phase interaction with the host cell (such as blood cell, such as leucocyte and blood platelet) in microenvironment during moving development With this may cause the detection of CTC and separate to have more challenge.

Summary of the invention

The present disclosure describes using the bound fraction that is combined with blood-platelet specific from sample fluid, such as biofluid, What such as blood, marrow, pleural effusion were related to blood platelet is separated in ascites has core target cell, and such as circulation epithelial cell, circulation is swollen The method of oncocyte (CTC), circulating endothelial cells (CEC), circulating stem cell (CSC), neutrophil leucocyte and macrophage and it is System.The method is included in any target cell such as CTC caused in bound fraction and sample, for example, coat hematoblastic CTC knots The sample fluid comprising blood platelet relevant target cells such as CTC is set to flow through certain room such as cell capture chamber to be formed under conditions of conjunction Compound；Then separated from these compounds and collect target cell, so as to separate target cell from sample fluid.

In some embodiments, using be designed as realizing blood platelet target the system such as stage that target cell captures or Two-stage microfluid system implements the method.In some embodiments, before sample fluid is passed through cell capture chamber, can With free, uncombined to remove first by microfluid debulk (debulking) device treatment fluid such as blood sample Blood platelet and red blood cell (RBC).Debulk device can include one or more micro-pillar arrays to realize based on hydrodynamics chi Very little sorting.Then target cell is contained obtained by being processed using the cell capture chamber using antiplatelet antibody functionalization The related target cell such as CTC of blood platelet is captured with high flux to the sample fluid of leucocyte (WBC).In some embodiments, Cell capture chamber can include that any blood platelet relevant target cells and platelet antibody phase interaction in sample fluid can be strengthened Mixed structure.In some embodiments, such mixed structure is presented with being referred to as the structure of " herringbone " micro-mixer.

Under normal conditions, this disclosure relates to separated from sample fluid as described in the present application blood platelet it is related have core The method that target cell for example circulates epithelial cell, CTC, CEC, CSC, neutrophil leucocyte and macrophage.The method includes obtaining Cell capture chamber, the cell capture chamber contains multiple bound fractions of one or more walls for being bound to room, the wherein joint portion Divide and combined with blood-platelet specific；The related bar for thering is core target cell to combine of any blood platelet in bound fraction and sample is made Sample fluid stream is set to cross cell capture chamber to form compound under part；And separated from compound and collect that blood platelet is related has Core target cell, so as to separate target cell from sample fluid.

In some embodiments, method described herein is additionally may included in makes sample fluid stream cross cell capture chamber Sample fluid is processed using platelet suppressant drug before, wherein platelet suppressant drug suppresses from adhering to not tying for other cells The blood platelet of conjunction.For example, other cells can be blood platelet, red blood cell and/or leucocyte.In various embodiments, blood Platelet inhibitor can be theophylline, adenosine, Dipyridamole, argatroban or prostacyclin I2.

In any implementation method described herein, bound fraction can be the antibody combined with blood-platelet specific.

In some embodiments, before the method is additionally may included in and sample fluid stream is crossed cell capture chamber, selection Property ground remove the uncombined blood platelet from sample fluid, while keep the blood platelet in sample fluid it is related have core target thin Born of the same parents.For example, promoting circulation of blood can be entered in comprising the passage containing micro-pillar array with the microfluidic device for performing certainty lateral displacement Platelet is eliminated.For example, microtrabeculae can be arranged into multirow, wherein microtrabeculae be expert at it is interior be spaced apart about 30 microns to about 60 microns away from From e.g., from about 35 microns to about 56 microns, subsequent row is spaced apart about 5 microns to about 15 microns of distance with previous row, such as Between about 5.6 microns to about 9.0 microns, and the microtrabeculae wherein in each subsequent row and the microtrabeculae in previous row laterally The distance of skew is expert at interior interval less than microtrabeculae.

In other embodiments, in microfluidic devices using centrifugal force or inertia force or both or by close Degree gradient centrifugation carries out blood platelet elimination.

In some embodiments, cell capture chamber and microfluidic device can be respectively positioned in single substrate or it can be with Connected in separate substrate and via catheter fluid.In some embodiments, cell capture chamber is contained within table The multiple herringbone structures arranged on face in sample fluid to produce micro swirl.

In some implementation methods for being designed for being selectively removed target cell, bound fraction be bound to comprising combine to One or more inner surfaces of the nanostructured of first component, wherein cell capture chamber be bound to using combine to multiple second The gelatin layer of component functionalization, and wherein nanostructured by combine to the first and second components binding interactions combine To the top layer of gelatin.In some implementation methods of these methods, the related core target cell that has of blood platelet passes through bound fraction knot Nanostructured is bonded to, and nanostructured separation blood platelet correlation is discharged from gelatin by melting gelatin at elevated temperatures There is core target cell.Or, can by gelatin layer apply partial cut stress or by using light target photo-thermal effect from Gelatin release nanostructured separates target cell.

In another aspect, this disclosure relates to separated from sample fluid blood platelet it is related have core target cell such as CTC System, such as two benches microfluid system.These systems include the first Room, second Room and by leading that two rooms fluidly connect Pipe.Especially, conduit fluidly connects the first Room product exit and the second chamber inlet.

In such systems, the first Room include have entrance, Waste outlet, the microchannel of product exit, and be arranged in into Mouthful and the micro-pillar array between outlet, wherein microtrabeculae arrangement embarks on journey and spaced a distance, and the distance is so that red blood cell and not With reference to blood platelet can flow through device reach Waste outlet and cause blood platelet it is related have core target cell horizontal by micro-pillar array To being moved to product exit, wherein between the microtrabeculae in each subsequent row and the microtrabeculae in previous row lateral shift away from Interval in being expert at less than microtrabeculae.

Second Room includes the microchannel with entrance and exit, and wherein fluid flow to outlet by microchannel from entrance, And be defined in its multiple groove for being arranged on the inner surface of one or more walls, base plate and the top board of microchannel of neutralization so as to Micro swirl is produced in the sample fluid；And be fixed to the bound fraction of at least one inner surface, wherein bound fraction with Blood-platelet specific is combined.For example, bound fraction can be the antibody combined with blood-platelet specific.

In the different embodiments of these systems, microtrabeculae be expert at it is interior be spaced apart about 30 microns to about 60 microns of distance, E.g., from about 35 microns to about 56 microns, and subsequent row is spaced apart about 5 microns to about 15 microns of distance with previous row, e.g., from about 5.6 microns to about 9.0 microns.In various embodiments, the first Room and second Room are respectively positioned in single substrate or it can Connected with separate substrate and via catheter fluid.

In some embodiments, groove in the second chamber includes tip and is connected to form two of V-arrangement with tip Arm, and groove is arranged into so that sample fluid flows to tip from arm.

In some implementation methods of these systems, bound fraction be bound to comprising combine to first component nano junction One or more inner surfaces of structure, wherein second Room be bound to using combine to multiple second component functionalizations gelatin layer, And wherein nanostructured by combine to the binding interactions of the first and second components be bound to the top layer of gelatin.

As used in this application, term " bound fraction " refers to adhere to target spot such as particle, molecule or cell Any molecule or reagent.Bound fraction includes such as ligand binding to, antibody, fit or nucleic acid molecules member.Some joint portions The dtex opposite sex is bound to target molecule or reagent, acceptor or cell surface marker thing such as on cell surface, and some non-spies The opposite sex is bound to the multiple target spots that can share common trait.

As used in this application, term " specific binding " refers to bound fraction in the sample comprising other particles or molecule In product selectivity and be preferably bonded to specific target spot such as particle, molecule or cell, be for example bound to the molecule on cell surface.

Compared with the existing CTC sorting technologies of the special biophysics of cancer cell and/or biochemical property are depended on, this Shen Please described method and system be conceived to active cell-cell interaction during cancer metastasis, it does not receive scale, cancer class The limitation of type or TSA expression.Importantly, this new method and system can separate it is very special There is core target cell subsets such as CTC, it is related to metastatic potential higher, it can be difficult to targetted by other technologies, and therefore It is early-stage cancer diagnosis and more preferably understands cancer how is spread there is provided valuable information.

Especially, to be that it is not relied on special on target cells for a benefit of herein described new method and system Property biomarker.Many current technologies depend on the expression of epithelial cell adhesion molecule (EpCAM), and this is one kind in the positive The biomarker of widely used targeting epithelial cell and CTC in system of selection.However, the expression of EpCAM is in clinical sample Between differ greatly and as epithelial-mesenchymal conversion (EMT) result its can also significantly be lowered with cancer progression.This Inventive method and system overcome this difficulty of existing method presence.

Unless otherwise defined, all technologies otherwise used herein and scientific terminology have and art of the present invention Those of ordinary skill's is generally understood that with identical meanings.Although can be by with described in this application those be similar or equivalent Method and material are used in implementation of the invention or test, but suitable method and material are as mentioned below.Carried in the application And all publications, patent application, patent and other bibliography are integrally incorporated by reference.If any conflict, with this theory Bright book and its included definition are defined.Additionally, material, method and embodiment are only exemplary and are not intended to limit. Details of one or more embodiments of the invention are listed in accompanying drawing and following description.Other features of the invention, purpose and Advantage will be obvious from specification and drawings and from claim.

Brief description of the drawings

Figure 1A is the schematic diagram via the related CTC metastases of blood platelet.

Figure 1B is to realize one signal of implementation method of two benches microfluidic platforms of blood platelet targeting CTC capture designs Figure.

Fig. 2A is one schematic diagram of implementation method of debulk chip.

Fig. 2 B are a schematic diagrames for implementation method of the micro-pillar array on debulk chip.

Fig. 3 is one schematic diagram of implementation method of micro swirl " herringbone " chip.

Fig. 4 A to Fig. 4 D are the flowings that particle such as cell or cell cluster flow through micro swirl " herringbone " chip shown in Fig. 3 A series of schematic diagrames of pattern.

Fig. 5 is to show the CTC from pulmonary metastasis patient blood samples for using EpCAM and CD41 antibody captures respectively The figure of counting.

Fig. 6 is to show individual cells, 1 or 2 WBC cluster and more than 2 figures of the CTC countings of WBC cluster forms.

Fig. 7 is to show the figure for adding that EDTA and prostacyclin I2 reduction Platelet-leukocyte aggregation are formed.

Fig. 8 A and Fig. 8 B are directed to unprocessed (8A) and process (8B) blood sample in microfluid " people through prostacyclin I2 The thermal map of the WBC captured on font " chip.

Same reference numerals represent similar elements in different figures.

Specific embodiment

Anticipate as shown in Figure 1A, it is believed that the interaction between tumour cell and blood platelet is played in haematogenous transfer Effect.Most noticeable evidence be found in several independent mice studies transfer and logical is suppressed by platelet consumption Cross platelet reconstitution and recover metastatic potential.Hematoblastic unique ability is activated and assembled to malignant cell for tumour on the surface Successful transfer provide many advantages, the process is referred to as the platelet aggregation (TCIPA) of tumor cell induction.Blood platelet may The vascular remodeling process in angiogenesis is not only facilitated, but also can be by making its escape shearing force and immunosurveillance notable The survival in blood of enhancing tumour cell.

According to new method described herein and system, the major class adhesion receptor on blood platelet can be used for from sample Separated in product fluid such as blood such as whole blood blood platelet it is related have a core target cell, such as epithelial cell, CTC, neutrophil leucocyte and Macrophage.Additionally, the research on signal transduction between blood platelet and tumour cell recently is disclosed from hematoblastic Growth factor/cell factor contributes to induction epithelial-mesenchymal conversion (EMT) and further enhances metastatic potential.Therefore, new method Can separate the CTC with enhanced metastatic potential with system, and thus may with its compared with other CTC that blood platelet is unrelated More preferable diagnostic message is provided.

The method that target cell-blood platelet cluster is separated from sample fluid

This method and system make use of interaction and profit unique between some karyocytes such as CTC and blood platelet The blood platelet for being combined with such target cell (be for example bound to as being coated on target cells) as generally existing based on The label targeting of cell simultaneously separates these target cells from sample fluid such as blood such as whole blood.

Under normal conditions, using the room containing multiple bound fractions, bound fraction is bound to of the room to new method Or multiple walls, wherein bound fraction combined with blood-platelet specific；Any blood platelet in bound fraction and sample is made is related Target cell makes the sample fluid containing target cell (if any) flow through the room to form compound under conditions of combining；And from compound Thing separates and collects target cell, so as to separate target cell from sample fluid.And, the method can also be comprised the following steps： Sample fluid is set to be selectively removed uncombined blood platelet and/or other contamination of cells before flowing through the room from sample fluid such as Red blood cell (RBC) in sample fluid while keeping the related target cell of blood platelet and/or being mixed to increase in the chamber Contact between blood platelet and bound fraction.

Because TCIPA is derived from the inherent general phenomenon for interacting between tumour cell and host's microenvironment, thus blood The method of platelet targeting has core target cell with the wide spectrum not relied in the detection of tumour film epitope and separation haematogenous transfer Potentiality.

For reduce WBC pollution and increase CTC purity, can before detection in blood sample add platelet suppressant drug with Stabilization blood.Can be used using various platelet suppressant drugs such as theophylline, adenosine, Dipyridamole, argatroban and prostacyclin I2 In stablizing blood.EDTA especially has with the combination of prostacyclin I2 in terms of Platelet-leukocyte aggregation thing (PLA) formation is suppressed Effect, it can make the quantity of pollution WBC in blood sample reduce 90%.

The bound fraction combined with blood platelet relevant target cells

Various bound fractions can be used to combine the blood platelet in sample fluid.For example, the different blood of targeting known to various The antibody of board surface acceptor, including CD41 and CD61 (subunit of integral protein α 2b β 3)；CD42b and CD42c (von The major receptors of the Willibrand factors (γ WF))；Glycoprotein VI (GP VI) (collagen receptor)；And TPO is received Body (TPO-R).These anti-CD41 antibody can be efficiently used, because it has capture efficiency higher and relatively low to blood platelet Non-specific binding.

Various methods such as silane chemistries can be used bound fraction functionalization in solid support such as PDMS or glass table On face.Different silane precursors with amine, aldehyde or sulfydryl end group can be handed over the PDMS/ glass surfaces processed through oxygen plasma Connection, it further can be conjugated from different bound fractions.In a particular example, the solid of microfluidic channel form is supported Thing is modified through 3-mercaptopropyi trimethoxy silane, is subsequently adding N-y- maleimidobutyryloxy succinimide ester conducts Joint, is finally conjugated with NeutrAvidin (neutravidin).Then any biotinylated bound fraction is such as biological The platelet antibody of elementization can by avidin-biotin easily functionalization on the device.

Sample fluid is set to flow through the room with bound fraction

After functionalisation of surfaces, the device is closed to avoid non-specific binding usually using reagent, such as using effective The bovine serum albumin(BSA) (BSA) of amount, such as 3%BSA.Then, designated volume such as 1 to 10mL, such as 2,3,4,5 or 6mL are made Sample fluid, such as blood, such as whole blood or buffy coat flow into the system.Generally at ambient temperature with set pressure example Such as 0.03 to 0.15psi, such as 0.05,0.075 or 0.1psi and 1-5mL/ hours, such as 1,2,3 or the stream of 4mL/ hours Speed treatment sample fluid.

Make sample fluid debulk to remove uncombined blood platelet

Due to there are a large amount of blood platelets (~10 in whole blood⁵/ μ L) and it has the latent effect for making bound fraction saturation, First blood sample can be processed to remove free, uncombined blood platelet.Additionally, removing some or most of red blood cells (RBC) it is favourable.Debulk can be realized using such as density gradient centrifugation or the cell separation based on microfluid.

Density gradient centrifugation is carried out usually using the blood sample being collected in CPT pipes, and anti-coagulants and polyester are contained in the pipe Gel and density gradient liquid.After centrifugation, then there is the cell of core by carefully suctioning out collection from liquid surface, wherein existing From red blood cell and hematoblastic micropollution.

The blood debulk based on microfluid can be realized using different designs well known in the art and technology.For example, can be with Bending for example snakelike microfluidic channel in utilize inertia force, with by centrifugation or inertia force (such as inertia focusing) or this Both difference based on its size are focused on and sorting cell (see, for example, U.S. Patent number 8,807,879).In addition it is also possible to sharp Various sizes of cell is sorted with hydrophobic filter and/or standing acoustic waves.

In another embodiment, it is possible to use it is small that microfluid debulk device removes uncombined blood from sample fluid Plate.Such device can be made up of one or more micro-pillar arrays, with realize based on hydrodynamic size sorting, such as it is following enter What one step was described in detail.Under normal conditions, these microfluid debulk devices use the sorting based on hydrodynamic size with reality The low sheraing microfluid debulk of existing whole blood.RBC, blood platelet and plasma protein are discarded, but retains karyocyte (WBC and CTC) And be in be handed to the second stage for carrying out target cell such as CTC captures.The operating principle of microfluid debulk is to be based on depending on The certainty lateral displacement of hydrodynamic size, wherein flow through micro-pillar array simultaneously containing cell and cell-free solution result in quickly Key dimension separation (see, for example, U.S. Patent number 8,986,966 and U.S. Patent number 8,585,971).Also referring to, Ozkumur etc., " Inertial Focusing for Tumor Antigen-Dependent and-Independent Sorting of Rare Circulating Tumor Cells,”Science Translational Medicine,5 (179):179ra47(DOI:10.1126/scitranslmed.3005616)(2013)。

(including the space between microtrabeculae or the displacement between space and adjacent lines) and flow velocity are configured by optimization array, The free of 5-log can be achieved over and be not associated with hematoblastic consumption, while keeping most of karyocyte.Then will can contain The fluid product for having core target cell and leucocyte (WBC) be introduced directly into as described in the present application contain blood-platelet specific combine Partial room.

Biased sample fluid is strengthening binding interactions

In order to strengthen the interaction of any blood platelet relevant target cells and bound fraction in room in sample fluid, Ke Yi Interior includes mixed structure.For example, can be used for including zigzag, snakelike or torturous channel different channel design designs passive mixed Close.Further, it is also possible to pass through introduce by acoustics, pressure disturbance or dielectrophoresis technology realize active microring array further enhance it is mixed Close performance.

A kind of useful design is referred to as " herringbone " micro-mixer, and it is included in being used for using platelet antibody functionalization High flux is captured and (see, for example, on one or more inwalls, base plate or the top board of the room of the related target cell of blood platelet such as CTC PCT Application No. WO 2010/036912).Under normal conditions, by 1-10mL sample fluids such as erythrocyte sedimentation rate brown layer or through debulk Blood flow into the system.Generally under 0.03-0.15psi such as 0.05,0.075 or 0.1psi under room temperature condition with 1 to The flow velocity of 2mL/hr makes the sample fluid of continuous shake pass through the device.

Process the target cell captured in room

The all cells captured in cell capture device can be processed to identify, for example, be surveyed using dyeing Fixed, the dyeing of such as four colors is determined, and is characterized with carrying out target cell identification and blood platelet simultaneously.Will be for tumor marker (for example EpCAM, cytokeratin, EGF-R ELISA (EGFR), human epidermal growth factor receptor 2 (HER-2), cadherin- 11 and 4', 6- diamidino -2-phenylindone (DAPI)) be positive capture cell, and optionally for hematopietic markers thing (such as CD45, CD14, CD16) also for those negative scorings are CTC.Count range from 0.4 to 8.5 CTC/mL whole blood can be used The new method of sample realizes reliable CTC captures.Other target cells can be identified using other labels.It is, for example possible to use CD24, CD133 and CD326 detect epithelial cell；Neutrophil leucocyte is detected using CD15, CD16 and CD66b；And use CD11b, CD68 and CD163 detect macrophage.In addition it is possible to use CD34 and CD146 identify circulating endothelial cells (CEC) with And identify circulating stem cell (CSC) using CD44, CD90 and ALDH1.

CTC is captured using the device of anti-epithelial marker thing antibody such as EpCAM antibody functionalization such as when using prior art When on microfluidic device, the consistent positive hit for reducing compared with the method for the present invention is as a result shown.Referring to Fig. 5, it is such as It is discussed in more detail in Examples below 2, shows that cell capture system described herein can be captured reliably and come from The unicellular or cell cluster shape of the metastatic cancer patient originated with epithelium (lung, mammary gland) or non-epithelium (melanoma) tumour The CTC of formula.Using this blood platelet targeted approach have CTC segment counts higher be due to the new method can from lung and its He is epithelial tumor type capture CTC, and above-mentioned tumor type may lose its epithelial character and therefore become more to be difficult with Anti- epithelial marker thing antibody such as EpCAM antibody targets.

EpCAM targeted approach before this generally produces CTC limitedly related to blood platelet, and uses described herein The CTC of new method capture shows the blood platelet covering of wide scope, including completely by the CTC's of blood platelet/fibrin cladding Special subgroup.The CTC that these blood platelets are covered is assumed to be high metastatic potential precursor, because blood platelet enhances tumour cell Survival and propagation, but it is difficult to be captured by the conventional positive selection method of target tumor surface antigen.Can be using fixed Amount imaging method further studies the correlation between CTC phenotypes and blood platelet distribution.With reference to receiving that our groups develop recently Control cell release strategy, it is also possible to realize the genotype research of individual cell level, this expection will further expand us in cancer Knowledge in terms of transfer.

Once capture the related CTC of blood platelet using new method, it is possible to use identify with lung cancer sample identical scheme Breast cancer CTC, and antibody or mixtures of antibodies the dyeing identification melanoma by using targeting melanoma specific antigen CTC (for example, any one or more in anti-CSPG4, MCAM, TYRP1 and α-SMA antibody).

Captured target cell is purified by selectivity release

In some sample fluids as being aged in whole blood, it is understood that there may be the related WBC of a large amount of blood platelets, it is to use new method Separated together with the target cell related to blood platelet.

In some embodiments, new method can include that selectivity release is thin with the target that the room containing bound fraction is combined The step of born of the same parents such as CTC.For example, as described in International Publication No. WO 2014/121204, cell capture chamber can be wrapped Include the bound fraction combined with nanostructured, nanostructured include combining in itself to first component, wherein the one of the room or Multiple inner surfaces be bound to using it is multiple combine to second component functionalization gelatin layer, and wherein the nanostructured passes through With reference to the binding interactions of the first and second components be bound to the top layer of gelatin.Then target cell is (by its correlation Blood platelet is bound to the bound fraction combined with nanostructured) can be discharged from room by the one kind in two kinds of releasing mechanisms.

In the first releasing mechanism, can be for example, by melted gelatin at elevated temperatures by nanostructured from gelatin Upper release separates and removes target cell such as CTC from cell capture chamber.By a liter high-temperature, such as more than 30 DEG C, such as 37 DEG C, institute The target cell of capture can discharge in a batch.Or, in second releasing mechanism, apply local by gelatin layer Shearing force, can be such that target cell is discharged from gelatin.By increasing local shear forces in gelatin, for example, filled by using vibration The power for applying control frequency is put, such as the micro- sharp device described in PCT WO 2014/121204 can be from cell capture chamber Selectivity release is unicellular.

In addition to single CTC (about 55%), new method also separates various sizes of cell cluster in blood platelet targeting platform. In fact, the CTC captured more than 40% is CTC/WBC cluster forms.Fig. 6 is seen, which show CTC and 1 or 2 of about 30% WBC clusters, and about 15% more than CTC and 2 WBC cluster.The quantity of interaction WBC is with the blood platelet around CTC Cover and increase.Current method target single CTC and CTC/WBC clusters ability (the latter be very difficult to by it is conventional based on The positive or negative system of selection of compatibility is separated) will make it possible to carry out cancer metastasis entirely by non-invasive blood biopsy Face characterizes, and it also opens new chance, such as CTC cultures, because can improve CTC in specific microenvironment in downstream application Survival/propagation.

The ability for separating CTC/WBC makes it possible to separate the cell mass that can be lost in other existing CTC isolation technics. For example, Solid phase CTC isolation technics is used in combination magnetic material targeting WBC using based on antibody.In these methods, will Magnetic material functionalization is so that it specifically binds with WBC and is introduced into fluid sample before microfluid system is entered.So Fluid sample undergoes the magnetic gradient of orientation to cause magnetic-particle and in connection any thin into " deflected channel " afterwards Born of the same parents deflect in a certain direction.Introduced when by the fluid sample containing the target cell/particle combined with one or more magnetic-particles During deflected channel, the magnetic force produced by neighbouring magnet along magnetic gradient direction drawing magnetic-particle (and accompanying cell/ Grain).In the case where Solid phase CTC is separated, this any other particle/cells turn for separating WBC and being attached to WBC " waste " passage is moved to, and separates the CTC being not associated with " product " passage.This method result in any CTC combined with WBC Loss, it is combined with magnetic-particle and is entered waste passage.

Two benches microfluidic platforms described herein do not exist drawbacks described above.

The microfluid system of target cell is separated from sample fluid

As described above, using cell for separating the related method for having core target cell such as CTC of blood platelet from sample fluid Capture chamber, cell capture chamber contains the multiple bound fractions combined with one or more walls of the room, base plate and/or top board, its Middle bound fraction is combined with blood-platelet specific.In the target cell knot for causing bound fraction related to any blood platelet in sample The fluid sample containing target cell is set to flow through the room to form compound under conditions of conjunction.The process cause target cell such as CTC from Separated in compound and collected, so as to separate target cell from sample fluid.And, as shown in fig. 1b, the system can be wrapped Include upstream components (first stage 110), its before sample fluid stream is crossed cell capture chamber (second stage 120), from sample Uncombined blood platelet and other cells such as red blood cell (RBC) are optionally removed in fluid, while in keeping sample fluid The related CTC of blood platelet.

As shown in fig. 1b, can be whole blood 130 to the input fluid of the system.The blood that first stage 110 will be not associated with Platelet and other cells such as RBC 140 are separated with karyocyte (150), and karyocyte (150) continues through the system and enters Enter blood platelet target capture room (120).

First stage --- remove red blood cell and hematoblastic debulk device from sample fluid

For the first stage, whole system can include debulk device 200, such as by one or more micro-pillar arrays The microfluidic device of composition, RBC and free blood platelet are removed will pass through the sorting based on hydrodynamic size from whole blood, It causes that less blood platelet and RBC leave the device as waste, while so that larger WBC and CTC and cell cluster enter Enter second stage, i.e. cell capture chamber.

As shown in Figure 2 A, first stage system can be produced on one chip 200.In some embodiments, core Piece can include entrance 202, one or more micro-pillar arrays 205 (being in detail in this figure two) and outlet 203.

As shown in Figure 2 B, the first stage in the system can use 205 points of cellifugal fluid forces of micro-pillar array Credit screening device, micro-pillar array 205 optionally passes through particle based on its size, shape or deformability.In micro-pillar array The size in space, shape or deformability are determined can be by the cell type of the array.Can be by two or more microtrabeculaes Array is connected or parallel arrangement, such as continuously removing the cell of size increments.For the description of this microfluid system, referring to Such as U.S. Patent number 8,986,966, U.S. Patent number 8,585,971 and Ozkumur etc. (2031), as described above.

The size of various microtrabeculaes 210, geometry and arrangement can be used in device described herein.Can be using not The microtrabeculae of similar shape, such as with the microtrabeculae of circle, square, rectangle, ellipse or triangular-section.Can be between microtrabeculae The size in space 220 and the shape of microtrabeculae optimize to ensure rapid and efficiently separate.For example, the size range of RBC is 5-8 μm of magnitude, and hematoblastic size range is 1-3 μm of magnitude.The size of all WBC is all higher than 10 μm.In view of these Size, although can increase the speed of RBC and blood platelet by array using the space between bigger microtrabeculae, increases empty The size of gap can also increase the risk of loss WBC.Less void size ensure that more effectively capture WBC, but can also subtract Slow RBC and hematoblastic through-rate.According to the type of application, it is possible to use different geometries.For this method, The micro post diameter that can be used is for about 10 to 30 μm, e.g., from about 15 to 24 μm, such as 10,12,15,17,20,22,24,25 or 27 μm.Space or interval between microtrabeculae are for about 10 to 40 μm, and such as 20 to 32 μm, such as 15,20,25,30,35 or 40 μm is have Effect.

Micro-pillar array can be produced using various methods.For example, can enter by substrate such as glass, metal or polymer Molding, electroforming, etching or drilling is gone to form micro-pillar array.It is, for example possible to use for example poly- (dimethyl of simple micro-fabrication technology Siloxanes) (PDMS) soft lithographic, polymer casting (for example using epoxy resin, acrylic resin or polyurethane), it is injection-molded, Polymer heat embossing, laser capture microdissection machining, film surface micromachining, deep etching, electroforming are carried out to glass and silicon And 3-D manufacturing technologies such as stereolithography manufactures the passage and micro-pillar array of herein described device.Big portion in these devices Divide and replicate micro- feature using photomask.

For the characteristic size more than 5 μm, it is possible to use the emulsion mask based on transparency.Characteristic size is 2 to 5 The chromium photomask based on glass may be needed between μm.For smaller feature, it is possible to use the electron beam based on glass Direct write mask.Then mask is used to be defined in the case of silicon or glass the photoresist pattern of etching or definition is replicated Former, such as, using SU-8 photoresists, then can be used as polymeric material such as PDMS, epoxy resin and acrylic acid The duplication molding mother matrix of resin.Then prefabricated passage can be connected in rigid basement such as glass to complete the device.Its His manufacture method is that device well known in the art and described herein can be by homogenous material or the combination manufacture of material.

Design and manufacture specific first stage hydrodynamics sorting core on silicon using deep reactive ion etching Piece.Microfluid debulk part is formed using anode combination glass cover encapsulating chip.Formed using the makrolon manifold of customization With fluidly connecting for substrate and second stage cell capture chamber.

Second stage --- cell capture chamber

In different embodiments, cell capture chamber can be use blood platelet bound fraction by one or more walls and/ Or the simple microfluidic channel of base plate functionalization, or can be finer and including mixed structure to strengthen and increase sample The quantity that blood platelet correlation CTC in product fluid is contacted with blood platelet bound fraction.

As shown in Figure 3, cell capture chamber can form " the people using the generation micro swirl of antiplatelet antibody functionalization Font " room (or " chip "), for the related CTC of high flux capture blood platelet (referring to PCT Application Publication WO 2010/ 036912).In this embodiment, the room forms the microchannel for being formed in microfluidic devices, wherein formation is extended into The groove (or projection) of the wall of (or leaving) microchannel, promotes what is suspended in a fluid to be produced in the fluid for flow through passage The flow pattern of the interphase interaction of the inner surface of arbitrary cell and conduit wall.Increased interaction can cause in the channel The increase of the CTC quantity of capture, and therefore cause total capture rate of microfluidic device to improve.In this embodiment, will The capture rate of microfluidic device is defined as the ratio of the CTC quantity and TCS for flowing through passage for capturing in the channel.

By adjust the architectural feature (including such as device base material, passage and groove size etc.) of microfluidic device with And fluid flow parameters (such as the flow rate based on grain type and the fluid type of the particle that has wherein been suspended) can be further Improve efficiency.

Fig. 3 describes the example of the such microfluidic device for using soft lithography to produce.As mentioned below, by micro- Form groove in one or more walls of passage, base plate and/or top board, by blood platelet bound fraction be coated on microchannel wall, On the inner surface of base plate and/or top board and make sample fluid flow through microchannel the related CTC of blood platelet is trapped in into cell to catch In obtaining the microchannel of room.

Fig. 3 shows the miniflow of the groove 335,340 of the upper wall (or top board) with the passage 315 for extending into device 300 Body device 300.In some embodiments, cell capture chamber includes the projection (for example, V-arrangement projection) stretched out from wall, and It is not the groove in the wall for extend into passage 315.In some embodiments, symmetrical grooves 335 include two arms, its respectively across The length crossed between first end 350 and summit 345 and second end 355 and summit 345.In exemplary implementation method In, the angle α between two arms is 90 °.In some embodiments, between two-arm the scope of angle α between 10 ° to 170 °. In some implementation methods, microfluidic device 300 can include the upper substrate 305 combined with lower substrate 310, and it can be using suitable Suitable material manufacture.For example, upper substrate 305 can use elastomer, such as dimethyl silicone polymer (PDMS), and lower base Bottom can be manufactured using glass, PDMS or other elastomers.

Or or in addition it is possible to use plastics-production substrate, such as polymethyl methacrylate (PMMA), makrolon, Cyclic olefine copolymer (COC) etc..Under normal conditions, the material of the upper substrate of selection manufacture and lower substrate can be readily produced example Such as be easy to etch and can provide be easy to test optical property for example can be optically transparent, and can be it is nontoxic, To cause for the cell being attached in substrate without negative effect.Additionally, the material is preferably without autofluorescence or with having The autofluorescence of limit.And, the material is readily able to Guan Nenghua so that analyte can be attached to substrate.Additionally, the material can With with mechanical strength, to provide intensity to microfluidic device 300.Upper substrate 305 can be fastened securely to lower substrate 310, microchannel is formed therebetween, as mentioned below.

In some embodiments, microchannel 315 can have includes two side walls 320 and 325 and in upper substrate 305 The rectangular cross section of the upper wall 330 of middle formation.Using the term such as "up" and "down" of relative position in order to describing and representing The necessary relative position of position in accompanying drawing rather than feature.For example, can be by the device orient with so that groove is located at passage Basal surface on or cause passage central axis upright extend.Or, the cross section of microchannel 315 can be several shapes In one kind, including but not limited to triangle, trapezoidal, semilune etc..Once being connected to substrate 305, lower substrate 310 can be with shape Into the lower wall of microchannel 315.In some embodiments, microchannel 315 is included in what is formed in the upper wall 330 of microchannel 315 Multiple grooves 335.Or, groove 335 can be formed in any wall, and/or can be in more than one wall of microchannel 315 Formed.Groove 335 can be across the whole length of wall, or across only a part for wall.

Fig. 4 A-4D are to show that particle suspension flows through microchannel with planomural and with the another of the groove formed in wall The schematic diagram of individual microchannel.Fig. 4 A show the microfluidic device 400 including the microchannel 405 with rectangular cross section.It is micro- logical The wall in road 405 do not include as described by for microfluidic device 300 the surface of groove, i.e. wall be flat.Shown in figure Flow through the particle suspension of the target cell 425 related containing the blood platelet being suspended in fluid of microchannel 405.And it is opposite It is that Fig. 4 B show the similar suspension for flowing through microfluidic device 300.

When fluid is flowed through by groove 335, and the herringbone pattern for being formed is formed a line in the microchannel 315, in fluid path In groove 335 destroy flow of fluid.In some embodiments, it is particularly for example recessed according to flow velocity and the size of groove Angle between the size and groove two-arm of groove, the destruction of fluid flow causes to generate micro swirl in a fluid.Produce Micro swirl is because groove causes fluid to be flowed along the direction of the principal direction (i.e. axial direction) transverse to flow of fluid.One In a little implementation methods, although not producing micro swirl, groove 335,340 is induction of enough destructions with changing section fluid Flow path, so as to increased the interaction between wall and particle.

When not having any groove, as shown in FIG. 4 C, the target cell 425 of the blood platelet correlation being suspended in fluid is with basic Upper linear form passes through flat microchannel 405 so that be only close to those of flow field edge (such as close to the wall of microchannel 405) Particle 425 may there occurs interaction with the antibody of the wall for being bound to microchannel 205.And by contrast, as shown in fig.4d, Flow path through the related target cell 425 of the blood platelet of herringbone grooves pattern is destroyed by the micro swirl in fluid, be increased The quantity of cell and the interphase interaction of the antibody for being bound to wall and/or groove.Micro swirl is subject in the upper of microfluidic device 300 The influence of the architectural feature of each groove 335 formed in wall 315.

The herringbone chip of second stage can be using soft lithography as previously mentioned by being bound to substrate of glass PDMS is made.Then anti-CD41 antibody (Abcam) can be used by chip surface using avidin-biotin Functionalization.

First stage hydrodynamics sorting chip and second stage herringbone chip can be prepared on one chip, or Single chip can be made into and connected by conduit, such as plastic tube or other pipes.

Embodiment

The present invention is described further in the following embodiments, and it is of the invention for what is described in the claims Scope is not construed as limiting.

Embodiment 1 --- CTC is separated from clinical sample

This method is tested to determine its efficiency for separating CTC from different types of cancer.

Advanced lung cancer, breast cancer and melanoma patients are recruited according to the scheme ratified through institutional review board (TRB). All specimen collections are extremely contained anti-coagulants EDTA's(Becton-Dickinson) in pipe, and in blood drawing 4 It is processed using micro-fluid chip in hour.Collect blood after add immediately extra platelet suppressant drug such as theophylline, Adenosine, Dipyridamole, argatroban and prostacyclin I2.Fixed blood sample is directly collected extremelyBCT In pipe (Streck).

Sample is run on two benches microfluid system mentioned above.Especially, deep reactive ion etching is used Manufacture first stage hydrodynamics sorts chip on silicon.Microfluid is formed using anode combination glass cover encapsulating chip Room.Use the makrolon manifold formation of customization and fluidly connecting for microchip.The space that we test between microtrabeculae is 20 Or 32 μm of two different array configurations.All of karyocyte is almost remained with the 20- μm of array in space and have Few pollution RBC, but its cell retention for will be greater than 21 μm and therefore may lose larger CTC or CTC clusters.And it is opposite , with the 32- μm of array in space for 8 to 30 μm between cell have extension opereating specification, but only remain 60% WBC.Granulocyte and the pouring of reported CTC sizes are less than by the cell lost in the 32- μm of array in space Bar cell, thus we select the array as the piece-rate system of CTC.

The herringbone chip of second stage is by being bound to substrate of glass using soft lithography as previously mentioned PDMS is made.Anti- CD41 antibody (Abcam) is used by chip surface functionalization using avidin-biotin.

As shown in Figure 5, it shows that (left side) uses EpCAM and CD41 antibody captures respectively to result in a graphical form From pulmonary metastasis patient 32 blood samples CTC count.Identified and blood platelet sign using CTC is carried out simultaneously The dyeing of four colors determines and all cells captured on chip is processed.Will for tumor marker (EpCAM, cadherin- 11) it is the positive with DAPI, while being that the negative cell score for being captured is CTC for hematopietic markers thing (CD45).At 32 21 in (66%) realize reliable CTC capture, its count range is from 0.4 to 8.5CTC/mL.

By contrast, the parallel CTC captures for carrying out show and hold on using the microfluidic device of EpCAM antibody functionalizations Continuous relatively low positive hit (Fig. 5, right side).It is because this method has that there is the method for blood platelet targeting CTC higher to count Capture may lose its epithelial character so as to be difficult to the ability by the lung CTC of EpCAM antibody targets.

Using CD61 antibodies on platelet dye and characterize its distribution around CTC surfaces.Caught using new system The CTC for obtaining shows the blood platelet covering of wide scope, including completely by the CTC subgroups of blood platelet/fibrin cladding.By this The CTC of a little blood platelet coverings is assumed to be high metastatic potential precursor, because blood platelet enhances survival and the propagation of tumour cell, but It is that it is difficult to be captured by the conventional positive selection method of target tumor surface antigen.Therefore, new system and method are capture These CTC provide new and improved technology.

Embodiment 2 --- the separation of different type cancer

Microfluidic platforms are extended so as to from originating with epithelium (mammary gland) and non-epithelium (melanoma) tumour CTC is separated in different type cancer patient.Mammary gland CTC is identified using the sample identical scheme with patients with lung cancer, but The mixtures of antibodies of targeting melanoma specific antigen (CSPG4, MCAM, TYRP1 and α-SMA) is used according to report before this Melanoma CTC is dyeed.Preliminary result shows that being respectively provided with reliable CTC for both cancer types captures (5 3 in breast samples, count range is from 0.9 to 2.7CTC/mL；5 in 6 melanoma samples, count range is from 1.4 To 17CTC/mL).Result with lung cancer is similar, and all CTC captured using current method are covered with different degrees of blood platelet Lid is related.

As discussed above, using EpCAM/ Epithelial-Cadherins 1 (green) as epithelium and a leaf label to lung and Mammary gland CTC is identified, but uses the mixture of CSPG4, MCAM, TYRP1 and α-SMA (green) antibody to melanoma CTC Dyeed.WBC and blood platelet are dyeed using CD45 (red) and CD61 (green) respectively.Microscope imaging is clearly Show the various types of CTC for being captured (result does not show).

Therefore, the system tested can be from originating with epithelium (lung, mammary gland) and non-epithelium (melanoma) tumour In metastatic cancer patient CTC is reliably captured in the form of individual cells or cell cluster.These results show that blood platelet is targetted CTC to capture to polytype cancer be effective.

Embodiment 3 --- anti-coagulants and CTC purity

Potentially a key issue of limitation blood platelet targeted approach performance is relatively low compared with other technologies CTC purity.Be observed on chip substantial amounts of pollution WBC (>10⁵/ mL), this causes that the analysis in downstream has challenge very much Property.The WBC for dyeing the most of capture of display using platelet-specific antibody is also coated by blood platelet.These be referred to as blood platelet- The formation of leukocyte recruitment thing (PLA) is derived from the expression of spontaneous platelet activation and subsequent p- selectins, and then it will It is bound to the PSGL-1 acceptors on leukocyte surface.PLA cannot be removed by the sorting based on size, and itself and blood platelet phase The CTC of pass is captured by antiplatelet antibody together.

In order to reduce the pollution of WBC and improve the purity of CTC, we test various blood platelets for stablizing blood and press down Preparation, such as theophylline, adenosine, Dipyridamole, argatroban and prostacyclin I2.Using the capture based on EpCAM as control with Assessment is per the WBC quantity in mL blood samples.Shown in the left side one of these results such as Fig. 7 arranges.Capture based on EpCAM is generally led Less WBC pollutions are caused, although this CTC separation methods have number of drawbacks discussed above.Suppressed using various blood platelets To being tested based on hematoblastic capture, as a result (solid rim represents untreated sample as shown in the right side one in Fig. 7 arranges for agent Product).It was found that EDTA effectively suppresses PLA with the combination (as shown in the open triangles in figure) of prostacyclin I2 being formed, and make dirt The quantity for contaminating WBC reduces by 90% (Fig. 7).

Formed to completely inhibit PLA, blood sample exists before treatmentIt is small 24 to be fixed in BCT pipes When, it is produced for the optimal CTC purity based on hematoblastic capture.These samples are referred to as " fixed blood " (hollow Circle).

Fig. 8 A and Fig. 8 B show and contain each WBC for being captured for mapping to its relative position on microfluidic device The thermal map of the microfluidic device of (stain).There is stronger pattern of attenuation in untreated blood sample, most of captured thin Born of the same parents are located at device portal, and this shows there is specificity interaction (figure by the blood platelet on surface between WBC and CD41 antibody 8A).And by contrast, the thermal map of blood sample processed through prostacyclin I2 shows significantly less capture cell, its with Machine is distributed in whole device (Fig. 8 B).

And, by using commercially available blood stablizing solution (such as Streck'sBCT is managed) it is soft fixed Blood sample, can suppress the PLA formed in most of processing procedure, because it causes blood platelet complete deactivation, so as to by CTC Purity substantially improve to suitable with conventional EpCAM target captures.In these researchs, CTC captures are unaffected, because TCIPA in vivo under the conditions of occur and the related aggregation of these blood platelets is formed before blood sampling.

The CTC for being designed for realizing blood platelet targeting is improved for this solution of blood platelet-WBC aggregations to catch The result of the two benches microfluidic platforms for obtaining.

Other embodiment

Have been described for multiple embodiments of the present invention.It will be appreciated, however, that not departing from the spirit and scope of the invention On the premise of can carry out various modifications.Therefore, other embodiment is within the scope of the appended claims.

Claims (29)

1. a kind of for separating the related method for having core target cell of blood platelet from sample fluid, methods described includes：

Cell capture chamber is obtained, the cell capture chamber contains multiple joint portions of one or more walls for being bound to the room Point, wherein the bound fraction is combined with blood-platelet specific；

Any blood platelet in making the bound fraction and the sample it is related have core target cell combine under conditions of make institute Sample fluid is stated to flow through the cell capture chamber to form compound；And

From the compound separate to collect blood platelet it is related have core target cell, so as to from the sample fluid separation blood Platelet it is related have core target cell.

2. method according to claim 1, wherein the blood platelet it is related have core target cell include circulation epithelial cell, Circulating tumor cell (CTC), circulating endothelial cells (CEC), circulating stem cell (CSC), neutrophil leucocyte and macrophage appoint One or more of meaning.

3. the method according to claim 1 or claim 2, wherein the sample fluid includes biofluid.

4. method as claimed in any of claims 1 to 3, methods described is additionally included in flows through the sample fluid The sample fluid is processed using platelet suppressant drug before the cell capture chamber, wherein the platelet suppressant drug suppresses not With reference to platelet adhesion reaction to other cells.

5. method according to claim 4, wherein described other cells include blood platelet, red blood cell and leucocyte.

6. the method according to any one in claim 4 and 5, wherein the platelet suppressant drug include theophylline, adenosine, Dipyridamole, argatroban or prostacyclin I2.

7. method as claimed in any of claims 1 to 6, wherein the bound fraction is included and blood-platelet specific With reference to antibody.

8. method as claimed in any of claims 1 to 7, methods described is additionally included in flows through the sample fluid Before the cell capture chamber, uncombined blood platelet is optionally removed from the sample fluid, while being maintained at described Blood platelet in sample fluid it is related have core target cell.

9. method according to claim 8, wherein blood platelet elimination is carried out in microfluidic devices, the microfluidic device Comprising the passage containing micro-pillar array performing certainty lateral displacement.

10. method according to claim 9, wherein the microtrabeculae is arranged into multirow, the interior interval wherein the microtrabeculae is expert at About 30 microns to about 60 microns of distance is opened, e.g., from about 35 microns to about 56 microns, it is micro- that subsequent row is spaced apart about 5 with previous row The distance that 15 microns of meter Zhi Yue, e.g., from about 5.6 microns to about 9.0 microns, and it is wherein described micro- in each subsequent row The distance of lateral shift is less than interval of the microtrabeculae in the row between post and the microtrabeculae in previous row.

11. methods according to claim 8, wherein in microfluidic devices using centrifugal force or inertia force or both Carry out the blood platelet elimination.

12. methods according to claim 8, wherein carrying out blood platelet elimination by density gradient centrifugation.

13. method according to any one in claim 9 to 11, wherein the cell capture chamber and microfluid dress Put and be respectively positioned in single substrate.

14. method according to any one in claim 9 to 11, wherein the cell capture chamber and microfluid dress Connected setting in separate substrate and via catheter fluid.

15. method according to any one in claim 1 to 14, wherein the cell capture chamber is contained within surface Multiple herringbone structures of upper arrangement in the sample fluid to produce micro swirl.

16. method according to any one in claim 1 to 15, wherein the bound fraction is bound to comprising with reference to right First component nanostructured, wherein one or more inner surfaces of the cell capture chamber are bound to using the combination pair Multiple second component functionalizations gelatin layer, and the first and second components that wherein described nanostructured passes through the combination pair Binding interactions be bound to the top layer of the gelatin.

17. methods according to claim 16, wherein the related core target cell that has of the blood platelet passes through the joint portion Divide and be bound to the nanostructured, and the nanometer is discharged from the gelatin by melting the gelatin at elevated temperatures Structure separate the blood platelet it is related have core target cell.

18. methods according to claim 16, wherein the related core target cell that has of the blood platelet passes through the joint portion Divide and be bound to the nanostructured, and received from described in gelatin release by institute's gelatin layer applying partial cut stress Rice structure separate the blood platelet it is related have core target cell.

19. methods according to claim 16, wherein the related core target cell that has of the blood platelet passes through the joint portion Divide and be bound to the nanostructured, and photo-thermal effect is targetted by using light and discharge the nanostructured separation from the gelatin The blood platelet it is related have core target cell.

The related core target cell that has of 20. method according to any one in claim 1 to 19, wherein blood platelet is circulation Tumour cell (" CTC ").

21. method according to any one in claim 3 to 20, wherein the biofluid includes blood, marrow, chest Chamber hydrops or ascites.

22. method according to any one in claim 3 to 20, wherein the biofluid includes blood.

23. is a kind of for separating the related two benches microfluid system for having core target cell of blood platelet, the system from sample fluid System includes：

First Room, including

With entrance, Waste outlet, product exit microchannel, and be arranged between the entrance and the outlet microtrabeculae battle array Row, wherein microtrabeculae arrangement is embarked on journey and spaced a distance, the distance enables red blood cell and uncombined blood platelet Described device is enough flowed through to reach Waste outlet and cause the core target cell that has of blood platelet correlation extremely to be produced by micro-pillar array lateral displacement Thing is exported, wherein the distance of lateral shift is less than between the microtrabeculae in each subsequent row and the microtrabeculae in previous row Interval of the microtrabeculae in the row.

Second Room, including

Microchannel with entrance and exit, wherein fluid flow to the outlet by the microchannel from the entrance, with And be defined in its multiple groove for being arranged on the inner surface of one or more walls, base plate and the top board of the microchannel of neutralization with Just micro swirl is produced in the sample fluid；And the bound fraction of at least one inner surface is fixed to, wherein the combination Part is combined with blood-platelet specific；And

First Room product exit is fluidly coupled to the conduit of second chamber inlet.

24. systems according to claim 23, wherein the microtrabeculae is expert at and interior is spaced apart about 30 microns to about 60 microns Distance, e.g., from about 35 microns to about 56 microns, and subsequent row is spaced apart about 5 microns to about 15 microns of distance with previous row, E.g., from about 5.6 microns to about 9.0 microns.

25. system according to any one in claim 23 and 24, wherein first Room and the equal position of the second Room In in single substrate.

26. system according to any one in claim 23 and 24, wherein first Room and the second Room are located at Connected in the substrate of separation and via the catheter fluid.

27. system according to any one in claim 23 to 26, wherein the groove include tip and with the point End is connected to form two arms of V-arrangement, and wherein described groove is arranged into so that the sample fluid is flowed to from the arm The tip.

28. system according to any one in claim 23 to 27, wherein the bound fraction is bound to comprising combination To first component nanostructured, wherein one or more inner surfaces of the cell capture chamber are bound to using the combination To multiple second component functionalizations gelatin layer, and the first and second structures that wherein described nanostructured passes through the combination pair The binding interactions of part are bound to the top layer of the gelatin.

29. system according to any one in claim 23 to 28, wherein the bound fraction is included and blood platelet spy The antibody that the opposite sex is combined.